Liposomes or lipid vesicles have been known since at least 1965. There are three general types of liposomes: multilamellar vesicles (MLV), onion-like structures having a series of substantially spherical shells formed of lipid bilayers interspersed with aqueous layers, ranging in diameter from about 0.1-4 .mu.m; large (greater than 1 .mu.m diameter) unilamellar vesicles (LUV) which have a lipid bilayer surrounding a large, unstructured aqueous phase; and small unilamellar vesicles (SUV) which are similar in structure to the LUV's except their diameters are less than 0.2 .mu.m. Because of the relatively large amount of lipid in the lipid bilayers of the MLV's, MLV's are considered best for encapsulation or transportation of lipophilic materials whereas the LUV's, because of their large aqueous/lipid volume ratio, are considered best for encapsulation of hydrophilic molecules, particularly macromolecules. SUV's have the advantage of small size, which allows relatively easy access to the cells of tissue, but their small volume limits delivery of hydrophilic aqueous materials to trace amounts. However, SUV's may be useful in the transportatio of lipophilic materials.
All of the early liposome studies used phospholipids as the lipid source for the bilayers. The reason for this choice was that phospholipids are the principal structural components of natural membranes. However, there are many problems using phospholipids for liposome-type structures. First, isolated phospholipids are subject to degradation by a large variety of enzymes. Second, the most easily available phospholipids are those from natural sources, e.g., egg yolk lecithin, which contain polyunsaturted acyl chains that are subject to autocatalyzed peroxidation. When peroxidation occurs, the liposome structure breaks down, causing premature release of encapsulated materials and the formation of toxic peroxidation byproducts. This problem can be avoided by hydrogenation but hydrogenation is an expensive process, thereby raising the cost of the starting materials. Cost is a third problem associated with the use of phospholipids on a large scale. A kilogram of egg yolk lecithin pure enough for liposome production, presently costs in excess of $40,000. This is much to high a cost for a starting material for most applications.
Because of the high cost and additional problems in using phospholipids, a number of groups have attempted to use synthetic amphiphiles in making lipid vesicles. For example, Vanlerberghe and others working for L'Oreal have used a series of synthetic polymers, primarily polyglycerol derivatives, as alternatives to the phospholipids. Similarly, Kelly and a group at Sandoz, Inc. have tried aliphatic lipids.
Recently, there has been some indication, particularly from the L'Oreal group, that commercially available surfactants might be used to form the lipid bilayer in liposome-like multilamellar lipid vesicles. Both surfactants and phospholipids are amphiphiles, having at least one lipophilic acyl or alkyl group attached to a hydrophilic head group. The hydrophilic head groups in the surfactants which have been tried include polyoxyethylene or polyglycerol derivatives. The head groups are attached to one or more lipophilic chains by ester or ether linkages. Commercially available surfactants include the BRIJ family of polyoxyethylene acyl ethers, the SPAN sorbitan alkyl esters, and the TWEEN polyoxyethylene sorbitan fatty acid esters, all avauilable from ICI Americas, Inc. of Wilmington, Del.
No matter what starting material is used to form the MLV's, substantially all of the methods of vesicle production reported in the literature use either the original Bangham method, as described in Bangham et al., J. Mol. Biol., 13:238-252 (1965), or some variation thereof. The basic approach followed starts with dissolving the lipids, together with any other lipophilic substances including cholesterol, in an organic solvent. The organic solvent is removed by evaporation using heat or by passing a stream of an inert gas (e.g., nitrogen) over the dissolved lipid to remove the solvent. The residue is then slowly hydrated with an aqueous phase, generally containing electrolytes and any hydrophilic biologically active materials, to form large multilamellar lipid membrane structures. In some variations, different types of particulate matter or structures have used during the evapoartion to assist in the formation of the lipid residue. The basis for these experiments are that by changing physical structure of the lipid residue, better vesicles may form upon hydration. Two recent review publications, Szoka and Papahdjopoulos, ann. Rev. Biophys. Bioeng. 9:467-508 (1980), and Dousset and Douste-Blazy (in Les Liposomes, Puisieux and Delattre, Editors, Tecniques et Documentation Lavoisier, Paris, pp.41-73 (1985)), summarize the methods which have been used tomake MLV's.
Once the MLV's are made, it is necessary to determine the effectiveness of the process. Two measurements commonly used to determine the effectiveness of encapsulation of biological materials in liposomes or lipid vesicles are the mass of substance encapsulated per unit mass of the lipid ("encapsulated mass") and captured volume.
The captured volume is the amount of solvent trapped within the vesicles. The captured volume is defined as the concentration of the aqueous fraction inside the vesicle divided by the concentration of lipid in the vesicle, normally given in ml/gm lipid.
Multilamellar lipid vesicles made using the classic methods have a low encapsulated mass for hydrophilic materials, normally in the order of 5-15%. In addition, the captured volume of solvent is normally in the order of 2-4 ml/g lipid. However, the encapsulated mass for lipophilic materials is much better in the multilamellar liposomes. Therefore, multilamellar liposomes made using the classical procedures are considered good for encapsulating lipophilic (hydrophobic) material but not hydrophilic.
The small unilamellar liposomes, which range is diameter from 20-50 nm, have a very low captured volume (approximately 0.5 ml/g) and also a very low encapsulated mass for hydrophilic materials (0.5-1%). However, since the lipid bilayer constitutes 50-87% of the total volume, these SUV's are excellent at transporting small quantities of lipohilic material. They also can be used to transport very small quantities of hydrophilic material to tissues where the MLV's or LUV's cannot reach.
Because of the problems in encapsulating large volumes and obtaining high encapsulated mass for hydrophilic materials, LUV's have been investigated. LUV's have large captured volumes (approximately 35 ml/gm lipid) and high encapsulated mass for hydrophilic materials (40-50%) but they are very poor in encapsulating hydrophibic or lipophilic materials. Because of these characteristics, LUV's are best suited to encapsulation of hydrophilic materials, including macromolecules. However, there are problems with the use of LUV's. Since there is only a single lipid bilayer surrounding a large aqueous center, the LUV's tend to be less stable then the other liposomes and more easily subject to degradation. Further, the low lipid/aqueous volume ratio makes it difficult to use LUV's for transport of any lipophilic materials.
Although there have been some experiments reported in the literature on using synthetic surfactants rather than phospholipids as a source for making multilamellar lipid vesicles, there are no reports showing any improvement in the ability to encapsulate either small or large hydrophilic molecules using these materials. In addition, there is no report of increased stability for lipid vesicles made with these materials. Therefore, the literature has given no indication that liposomes manufactured with these synthetic materials will be useful to achieve the hydrophilic and macromolecule delivery objects sought in the liposome field.
A further problem associated with multilamellar lipid vesicles (including the small unilamellar vesicles which are normally manufactured by sonication of the multilamellar vesicles) manufactured using standard methods is that these current processes are both slow and relatively inefficient in terms of material. For example, the standard time to manufacture multilamellar lipid vesicles is in the order 2-20 hours. If SUV's are required, the sonication which breaks the multilamellar lipid structures into SUV's takes additional time. This slow processing is unwieldy and expensive for any large scale use of lipid vesicles.
Accordingly, it is an object of the invention to provide a rapid and efficient process for the formation of multilamellar vesicles.
It is a further object of the invention to develop multilamellar vesicles with high encapsulated mass for hydrophilic materials and high captured volume.
It is another object of the invention to form lipid membrane structures without the use of organic solvents or detergents.
It is still a further object of the invention to provide a method for the rapid, efficient encapsulation of biologically active macromolecules into vesicles made of relatively inexpensive, readily available surfactants.
These and other objects and features of the invention will be apparent from the detailed description and the claims.